Normatividad Ambiental Vigente Al Subsector Avicola

Nine of the 39 clones identified with restricted expression patterns encode molecules that are identical or closely related to proteins with previously characterised expression, or functions in tissues other than early embryonic neural tissue. These cover a range of molecules including: (/) enzymes, (//) intracellular

components of signalling cascades, {Hi) membrane bound receptors, (tv) cytoskeleton

associated proteins, and (v) specific inhibitors of other proteins (Figure 3.1 and Figure 3.2).

(/) Segmentally expressed enzymes

Four clones encode different types of enzymes (0A2, O A ll, 3F10 and 4B1). Clone 0A2 is expressed throughout the hindbrain except for r5 at varying levels in different rhombomeres and sequence analysis shows it is identical to the chicken

brain-type creatine kinase {B-CK,) gene (Prichard, et a l, 1983). The B-CK protein is the brain isoenzyme of creatine kinase and is localised to the nucleoplasm; being highly expressed in the nucleus of proliferating astrocytes suggestive of a role in cell division/nuclear division (Manos and Bryan, 1993). The B-CK enzyme is involved in

energy metabolism transferring high-energy phosphoryl groups from creatine phosphate to the nucleoside diphosphate adenosine diphosphate (ADP), generating

adenosine triphosphate (ATP) (Hossle, et a l, 1986). B-CK is found in a number of

mature tissues and has a documented role during late rat brain development, being expressed during astrocyte cell divisions and myelination (Manos and Bryan, 1993).

Clone O A ll is expressed strongly in r5, and nucleotide sequence analysis suggests that it is a serine/threonine kinase highly related to the haem-regulated eukaryotic initiation factor subunit-2 alpha (eIF-2a) kinase (haem-regulated

inhibitor, HRI: Chen, et a l, 1991; Berlanga, et a l, 1998). Within erythroid tissues,

the HRI enzyme modulates the synthesis of globin with hemin and iron availability

(Chen, et a l, 1991). Subsequent sequencing of a full length clone corresponding to

O A ll (Genbank accession number AJF330008) has confirmed that it is highly related to, yet divergent from, HRI (Appendix C) as well as two other eIF 2a kinases:

interferon-inducible double-stranded RNA dependent protein kinase (PKR: Meurs, et

a l, 1990) and pancreatic eIF-2a kinase (PEK: Shi, et a l, 1998). Interestingly, the

catalytic domain (a four amino acid motif: Chen, et a l, 1991) is conserved between

O A ll and HRI (Appendix C). All three of these kinases modulate mRNA translation in eukaryotes by phosphorylating the eIF-2a component of the translational initiation complex. This prevents binding of the initiator methionine transfer RNA (Met- tRNAj) to the 40S ribosomal subunit and thus inhibits the initiation of protein

translation (Kimball, et a l, 1999).

Clone 3F10 is expressed throughout the midbrain and hindbrain with highest expression in rl and exhibits moderate amino acid similarity to the carboxyl terminus

of spermidine synthase (SPDSY) (Appendix C: Myohanen, et a l , 1994). The

SPDSY enzyme is part of the polyamine biosynthetic pathway and is required for the

production of spermidine from its precursor putrescine (Korhonen, et a l, 1995). It is

known that polyamines have a role in the regulation of cell growth and differentiation, but despite the research undertaken and its relative simple structure,

little is known about spermidine’s mode of action (Janne, et a l, 1991).

Clone 4B1 is expressed in the midbrain, hindbrain (r4 to r6) and in neural crest cells emigrating from r4, albeit at lower levels. In addition, 4B1 exhibits high sequence similarity with the dihydropyramidinase-related protein ULIP-4 (unc-33 Like Phosphoproteins), an amidohydrolase related to Urease (Holm and Sander,

1997). The C. elegans unc-33 gene product is an intracellular protein involved in the

control of neuritic outgrowth and axonal guidance (Hedgecock, et al., 1985).

Determination of the entire sequence of clone 4B1 (Genbank accession number AF330010) shows that it is very highly related to the amino terminal half of ULIP-4 (Appendix C).

{ii) Components of intracellular signal transduction cascades

Two clones represent intracellular components of signal transduction pathways (0A7 and OAIO). Clone 0A7 is expressed at high levels in the midbrain and r5 at HH stage 11-, and within r5 expression is restricted to the ventral two-thirds of the segment (Figure 3.1). Sequence analysis shows that clone 0A7 encodes for a peptide identical to the chick cellular inhibitor of apoptosis-1 protein (ch-lA Pl;

(You, et al., 1997). Ch-IAPl is recruited and binds to type 1 or type 2 tumour

necrosis factor alpha (TNFa) receptor after TN Fa ligand binding (Shu, et al., 1996).

This interaction is required to suppress the transcriptional activation of initiator

caspases (Wang, et al., 1998) and ch-IAPl can also bind directly to certain effector

caspases (Roy, et a l, 1997), both resulting in an inhibition of apoptosis (Deveraux

and Reed, 1999).

Clone OAIO is expressed at low levels in r3 and r5; initial sequence analysis showed that OAIO is most similar to JC310 (Appendix C). The human JC310 gene

(human Sinl), when overexpressed in Saccharomyces cerevisiae (budding yeast), is

capable of rescuing a temperature sensitive RAS''^"’ mutation, suggesting that Sinl

interacts directly with RAS proteins (Colicelli, et a l, 1991). Complete sequencing of

OAIO identified it as the chicken homologue of Sinl (Styl interacting protein), a

Schizosaccharomyces pombe (fission yeast) protein that interacts with Styl (also

known as Spcl or pH H l; (Degols, et a l, 1996). Styl is a mitogen-activated protein

kinase (MAPK), an intermediate in the stress-activated MAP signalling pathway that is required for the induction of mitosis and cell survival in response to changes in extracellular conditions (review see; (Marshall, 1994; Treisman, 1996). The Sinl protein is phosphorylated following stress in a Styl independent manner and is required for stress-dependent transcription via A tfl, the substrate of S tyl/S pcl

(Wilkinson, et a l, 1999). Clone OAIO is functionally equivalent to yeast Sinl as

(Wilkinson, et a l, 1999). This suggests that chick OAIO may be required for the propagation of MAPK signalling.

{iii) Transmembrane proteins

Clone 4F11 is expressed in specific dorsal cells posterior to r6 at HH stage 11, and is identical to zebra-finch transmembrane G-protein coupled central

cannabinoid receptor-1 (CBl; (Chakrabarti, et a l, 1995). Cannabinoid anandamide,

the psychoactive ingredient of Cannabis sativa, binds to and activates the CB l

receptor (Abood, et a i, 1997). Activated cannabinoid receptors transduce signals

across the membrane stimulating signal transduction pathways that regulate

adenylate cyclase activity, Ca^^ and channels and the mitogen-activated protein

kinase (MAPK) pathway (review see; (Howlett, 1998). Mice with targeted

disruptions in the CBl gene are viable but display reduced locomotor activity and

hypoalgesia (Ledent, et al., 1999; Zimmer, et a l, 1999).

(iv) Cytoskeleton associated proteins

At HH stage 10, clone GAS is expressed strongly in r2, r4 and also in migratory r4 neural crest cells and its sequence is highly similar to doublecortin.

Mutations in human doublecortin result in X-Linked Laminar Heterotopia and

Lissencephaly syndrome, a disease that is characterised by abnormal cortical lamination in the cerebral cortex. The disruption of the normal six-layered neocortex

is due to the abnormal migration of cortical neurons (des Portes, et a l, 1998;

Gleeson, et a l, 1998). The doublecortin protein has been shown to directly bind and

stabilise microtubules via interactions with tubulin, and is expressed widely in

migrating neurons (Francis, et a l, 1999; Horesh, et a l, 1999; Gleeson, et a l, 1999;

Taylor, et a l, 2000). Complete sequencing of the 0A8 clone (Genbank accession

number AF330009) confirmed that it is the chick homologue of doublecortin

(Appendix C).

(v) Specific antagonists of other proteins

Clone IDIO, expressed in the fore-, mid- and hindbrain at HH stage 11-, shares moderate amino acid similarity to the amino terminus of human

1988). RAI has previously been isolated from adult cerebrum and inhibits both

secretory and non-secretory type ribonucleases (Nadano, et a l, 1994). RNase activity

within a cell is regulated by the formation of RNase/inhibitor complexes and the ratio of RNase to RAI is high in proliferating tissue yet low during catabolic metabolism; the ration of RNase to RAI regulates mRNA turnover and thus protein

synthesis (Quirin-Stricker, et a l, 1968).